Asymmetrically dividing DM NBs do not express Prospero. Confocal images of NB divisions in a canonical NB lineage (top panels) compared to a DM lineage (bottom panels). Shown are representative CD8::GFP-labeled clones (green), seen around the NB in late larval brains stained for Miranda (MIRA, blue) and Prospero (PROS, red). Single channels are also shown in gray scale for better contrast. (a, b) Miranda forms cortical crescents at metaphase in both non-DM and DM NBs (asterisk). (c, d) Following asymmetric division, Miranda segregates into the small daughter cell and remains associated at high levels at the cortex soon after cytokinesis (the small newborn daughter cell is marked by an asterisk). Prospero co-localizes with Miranda in the dividing non-DM NBs (a, c, asterisks) and is nuclear in the oldest GMCs, which retain a low level of Miranda at the cortex (a, c, arrowheads), and in all other post-mitotic cells in the clone. In the DM NBs, Prospero is undetectable during mitosis (b, d, asterisks). (Note in (d) a canonical NB outside the clone (magenta asterisk) that shows co-localization of Miranda and Prospero and serves as internal control.) Recently born NB daughter cells show weak uniform cortical Miranda and lack Prospero (white dots in b). Polarized cortical Miranda during mitosis identifies these cells as IPs (b, arrows) and co-localization with Prospero is once again observed in these cells (b, insets). Cells with GMC-like (arrowheads) or neuronal expression of the markers are also observed as in canonical non-NB lineages. Scale bars: 10 μm.